Enzyme-substrate interactions play a critical role in a variety of biochemical processes. For example, these interactions play a role in the extracellular and intracellular signaling events and in the transcriptional and translational control of gene expression. Abnormal or disease states can be the direct result of abnormal enzyme-substrate interactions. Identification of specific enzyme-substrate complexes will enable the development of tools useful in the regulation of cell signaling and gene expression. This in turn will enable the biological manipulation of cell signaling and gene expression in the laboratory, as well as the development of therapeutic tools for manipulating cellular processes.
Enzyme-substrate interactions have been studied using both biochemical and genetic methods to gain understanding about enzyme-substrate binding activity and to identify unknown enzyme binding partners. These methods include coimmunoprecipitation, pull-down, chemical crosslinking, label transfer, tandem affinity purification (TAP), and two-hybrid assays (see Golemis, 2002, Protein-protein Interactions: A Molecular Cloning Manual, Cold Spring Harbor Laboratory and Fu, 2004, Protein-protein Interactions: Methods and Applications, Humana Press). The utility and applicability of the results obtained using these standard methods is limited with respect to obtaining a detailed understanding of enzyme-substrate binding activity, since the methods reveal which binding interactions occur in vitro or in an otherwise artificial environment. To truly understand the dynamics of enzyme-substrate interactions, it is first necessary to possess the ability to identify the interactions in vivo.
What is needed is a methodology that provides for the identification of enzymes that interact in vivo with a target substrate. Therefore, there exists a long felt need to provide a way to identify enzymes that interact with a pre-selected substrate in vivo. The present invention addresses and meets this need.